HER2 is an oncogenic receptor tyrosine kinase (RTK). It is overexpressed in about 20% breast cancer (BC) due to gene amplification, known as HER2-positive BC (HER2+ BC). Several HER2 inhibitors are available clinically and have significantly improved disease outcome. However, primary and acquired drug resistance is common. Most patients with advanced disease show disease progression after some time on treatment. Drug resistance is a major unresolved problem in HER2+ BC, and our long-term goal is to find a solution to this problem. In this project, we propose to investigate a recombinant human protein, i.e., PEPDG278D, for overcoming drug resistance in HER2+ BC. PEPDG278D is an enzymatically inactive mutant of peptidase D (also known as prolidase). Exogenously-administered PEPDG278D binds to HER2 and its family member EGFR, and in cancer cells overexpressing the RTKs, PEPDG278D disrupts their signaling units, directs them for lysosomal degradation, and inhibits the growth of the cells in vitro and in vivo. PEPDG278D inhibits HER2+ BC cells that are resistant to current HER2 inhibitors in vitro and in vivo. Yet, PEPDG278D is well tolerated in mouse studies and shows little effect on HER2 and EGFR in normal tissues where expression of the RTKs is very low. Cancer cells lacking HER2 and EGFR are insensitive to PEPDG278D as well. The objectives of this proposal are: 1) to determine the therapeutic activity and mechanism of action of PEPDG278D in HER2+ BC, and 2) to assess PEPDG278D safety and to understand how PEPDG278D spares HER2 and EGFR in normal cells. The central hypothesis is that PEPDG278D targets HER2 and EGFR specifically and its unique binding mode enables it to target overexpressed HER2 and EGFR strongly and selectively, thereby inhibiting drug-resistant HER2+ BC without causing toxicity. The rationale for the proposal is that completion of the research may propel PEPDG278D into clinical evaluation. We propose three specific aims to test the hypothesis: 1) to elucidate the target specificity of PEPDG278D, 2) to assess its therapeutic activity and mechanism of action, and 3) to determine its target selectivity and how it spares HER2 and EGFR in normal cells. An innovative combination of experimental methods will be used, including but not limited to isogenic cells, cells and tumors carrying clinically verified molecular changes that confer resistance to current HER2 inhibitors, primary normal human cells and humanized mice. The proposed research is significant, because it addresses a major problem in HER2+ BC, i.e., drug resistance. Expected outcome of this work includes: 1) showing that HER2 and EGFR are the sole therapeutic targets of PEPDG278D; 2) showing that PEPDG278D inhibits HER2+ BC resistant to current HER2 inhibitors and the underlying mechanisms; 3) showing that HER2 remains a critical therapeutic target in drug-resistant HER2+ BC; and 4) showing that PEPDG278D is non-toxic to normal cells and tissues and understanding the molecular basis. Our findings will have an important positive impact, because they will generate strong enthusiasm for clinical study of PEPDG278D.